Spreading depression (SD) is a common pathological process in the brain shown as propagating neuronal depolarization followed by activity depression over the brain, and it is closely related to migraines and epilepsy. Although O2 is known to fluctuate during SD, the difference of O2 responses at different sites in the same brain region remains unknown. In this study, we develop an in vivo electrochemical method with microelectrode arrays (MEAs) to monitor, in real time, O2 fluctuation at multiple sites of rat cortex during SD with high spatial/temporal resolution. Platinum nanoparticles are electrochemically deposited on the multiplexed electrodes of the MEAs to monitor O2 fluctuation simultaneously and selectively via a four-electron reduction process. Configuration of electrode arrays is designed rationally to exclude the probable crosstalk between neighbor recording electrodes during simultaneous measurements. With the MEAs, we find both the basal O2 levels and O2 fluctuations at different sites of the cortex during SD exhibit significant differences, indicating the intensity of energy metabolism and oxidative stress vary at different sites even in the same brain region. Further studies prove that O2 fluctuation is mostly caused by the increase of brain blood flow and the consumption of neuronal O2 during SD. Our study reveals that energy metabolism varies at different sites in brain cortex during SD propagation, which may provide new understanding for SD-related pathological processes.